Patria E. Danielson

Transgenic mice expressing β-galactosidase in mature neurons under neuron-specific enolase promoter control

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimers disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA® to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells‐2 (TREM‐2) was among the mRNAs identified as being expressed by unactivated microglia, but down‐regulated by lipopolysaccharide/interferon γ. In the healthy CNS, not all microglia expressed TREM‐2. Microglial expression of TREM‐2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood–brain barrier had the lowest percentages of TREM‐2‐ expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM‐2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM‐2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.

Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system

Fatty acid amide hydrolase (FAAH) is a membrane‐bound enzyme activity that degrades neuromodulatory fatty acid amides, including oleamide and anandamide. A single 2.5‐kb FAAH mRNA is distributed throughout the rat CNS and accumulates progressively between embryonic day 14 and postnatal day 10, remains high until postnatal day 30, then decreases into adulthood. FAAH enzymatic activity, as measured in dissected brain regions, was well correlated with the distribution of its messenger RNA. In situ hybridization revealed profound distribution of FAAH mRNA in neuronal cells throughout the CNS. The most prominent signals were detected in the neocortex, hippocampal formation, amygdala, and cerebellum. The FAAH distribution in the CNS suggests that degradation of neuromodulatory fatty acid amides at their sites of action influences their effects on sleep, euphoria, and analgesia. J. Neurosci. Res. 50:1047–1052, 1997. © 1997 Wiley‐Liss, Inc.

No hypothermic response to serotonin in 5-HT7 receptor knockout mice

With data from recently available selective antagonists for the 5-HT7 receptor, it has been hypothesized that 5-hydroxytryptamine (5-HT)-induced hypothermia is mediated by the 5-HT7 receptor, an effect previously attributed to other receptor subtypes. It has been established that the biologically active lipid oleamide allosterically interacts with the 5-HT7 receptor to regulate its transmission. The most well characterized effects of oleamide administration are induction of sleep and hypothermia. Here, we demonstrate, by using mice lacking the 5-HT7 receptor, that 5-HT-induced hypothermia is mediated by the 5-HT7 receptor. Both 5-HT and 5-carboxamidotryptamine, a 5-HT1 and 5-HT7 receptor agonist, in physiological doses fail to induce hypothermia in 5-HT7 knockout mice. In contrast, oleamide was equally effective in inducing hypothermia in mice lacking the 5-HT7 receptors as in wild-type mice. When administered together, 5-HT and oleamide showed additive or greater than additive effects in reducing body temperature. Taken together, the results show that 5-HT-induced hypothermia is mediated by the 5-HT7 receptor, and that oleamide may act through an independent mechanism as well as at an allosteric 5-HT7 receptor site to regulate body temperature.

The human retinal degeneration slow (RDS) gene: Chromosome assignment and structure of the mRNA ☆

Retinal degeneration slow (rds) is a mouse neurological mutation that is characterized phenotypically by abnormal development of rod and cone photoreceptors followed by their slow degeneration. This phenotype resembles the pathologic abnormalities seen in retinitis pigmentosa. The mouse rds gene has recently been cloned. Here we present the sequence of a full-length cDNA clone of the human RDS mRNA. We show that in human retina there are two RDS transcripts of 3.0 and 5.5 kb. By analysis of DNA from a panel of human X hamster somatic cell hybrids, and by direct in situ hybridization, we show that the RDS gene is located on the proximal short arm of human chromosome 6. Finally, we present information on the frequency of several observed restriction fragment length polymorphisms using the RDS cDNA. This information is of potential value for testing linkage of the RDS gene to the disease phenotype in families with retinitis pigmentosa.

Rhes: A striatal-specific Ras homolog related to Dexras1.

We have characterized an apparently full‐length cDNA corresponding to a rat mRNA, SE6C, previously identified by subtractive hybridization as being expressed predominantly in the striatal region of the brain. The SE6C mRNA encodes a 266 amino acid protein with significant similarity to members of the Ras‐like GTP‐binding protein family; thus, we have chosen the name Rhes, for Ras homolog enriched in striatum. The human homolog was found in a genomic sequence from human chromosome 22q13.1 and shares 95% identity with rat Rhes. Among the family of small G‐proteins, Rhes shares 62% identity with Dexras1, a mouse dexamethasone‐inducible Ras‐like protein. Both Rhes and Dexras1 have substantially longer C‐termini than other members of the Ras‐like small G‐protein family. Divergence between the C‐terminal sequences of Rhes and Dexras1 suggests that, although their functions are probably similar, they have unique properties. Bacterially expressed Rhes binds GTP, suggesting that the protein indeed has GTPase functionality. Although Rhes was not induced by dexamethasone, its full expression is dependent upon thyroid hormone availability. Its accumulation is postnatal, consistent with the dependence upon thyroid hormone. It is noteworthy that most striatum‐“specific” mRNAs characterized to date encode components of signal transduction cascades. J. Neurosci. Res. 57:782–788, 1999.

Differential gene expression in LPS/IFNγ activated microglia and macrophages: in vitro versus in vivo

Two different macrophage populations contribute to CNS neuroinflammation: CNS‐resident microglia and CNS‐infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN)γ‐treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2‐fold to 250‐fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFNγ‐injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the ‘microglial’ molecules (such as the tolerance associated transcript, Tmem176b) in CNS‐infiltrating macrophages. Bioinformatic analysis of the ∼19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFNγ‐activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS‐infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS‐resident microglia are heterogeneous and thus a universal microglia‐specific marker may not exist; (ii) the CNS micro‐environment plays significant roles in determining the phenotypes of both CNS‐resident microglia and CNS‐infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti‐inflammatory molecules.

The 5-HT5A serotonin receptor is expressed predominantly by astrocytes in which it inhibits cAMP accumulation : A mechanism for neuronal suppression of reactive astrocytes

The mRNA for the 5‐hydroxytryptamine receptor 5‐HT5A was detected at embryonic day 18 in the rat central nervous system and peaked by postnatal day 20. At all time points examined, 5‐HT5A immunoreactivity observed on astrocyte cell bodies and in the stellate processes not only colocalized with the astrocyte‐specific marker glial fibrillary acidic protein (GFAP) but was coordinately regulated with GFAP, increasing during development and during gliosis. Transfection of 5‐HT5A into glioma cells prevented the 5‐HT‐induced increase in cAMP observed in untransfected cells and decreased the relative forskolin response by approximately 20%, suggesting that the 5‐HT5A receptor couples negatively to adenylyl cyclase in astrocytes. Together, these results indicate a neuron‐to‐astrocyte serotonergic signaling pathway mediating cAMP concentrations, which could provide a neuronally driven mechanism for regulating astrocyte physiology with relevance to gliosis.

RGS9: A regulator of G-protein signalling with specific expression in rat and mouse striatum

A clone of the regulator of G‐protein signalling, RGS9, was isolated from a rat striatum‐minus‐cerebellum‐minus‐hippocampus subtracted library generated by directional tag polymerase chain reaction subtraction. The full‐length cDNA clone encodes a 444 amino acid protein containing an 118 amino acid RGS domain, which corresponds to an evolutionarily conserved domain that is present in all members of the RGS family of proteins. Outside of the homology domain, RGS9 shows more extended similarity to human RGS6 and RGS7, rat RGS12, and the C. elegans protein EGL‐10. During embryonic and early postnatal stages of development, two RGS9 transcripts of approximately 1.4 Kb and 1.8 Kb were detected in whole brain. After postnatal day 10, accumulation of the larger transcript increased progressively until adulthood at the expense of the smaller transcript, which was undetectable in the adult. In adult rat brain, the 1.8‐Kb RGS9 transcript was detected in the striatum but not in other brain regions or peripheral tissues. In situ hybridization in rat and mouse demonstrates that RGS9 mRNA is expressed predominantly in medium‐sized, spiny neurons of the neostriatum and in neurons of the nucleus accumbens and olfactory tubercle. Relatively strong signals were also detected in some hypothalamic nuclei. Its selective expression suggests that RGS9 may play an important role in modulation of the complex signalling pathways of the basal ganglia. J. Neurosci. Res. 52:118–124, 1998.

Nucleotide sequence and cellular distribution of rat chromogranin B (Secretogranin I) mRNA in the neuroendocrine system

The mRNA of rat secretory-vesicle protein chromogranin B is abundant in brain, adrenal medulla, and anterior pituitary. The primary translation product predicted from the cDNA sequence of this 2,337-nucleotide transcript corresponds to a hydrophilic 655-residue protein preceded by a signal peptide. Both termini of the mature 75-kD protein show extensive similarity to other chromogranins; the more variable internal region is characterized by glutamic acid clusters and numerous pairs of basic residues. In rodent brain, mRNA accumulation starts around embryonic days 13–14 and peaks by postnatal day 20. In situ hybridization in brain sections shows that the mRNA is enriched in the hippocampal formation, the endocrine hypothalamus, the olfactory system, and in anatomically distinct structures in the pons-medulla.